Acanthopanax acid polysaccharide with intestinal protection function and preparation method and application thereof

Abstract

The application discloses a kind of acanthopanax acid polysaccharides with intestinal protection function and its preparation method and application, belong to the field of functional food and biotechnology.The acanthopanax acid polysaccharide is by water extraction alcohol precipitation, after impurity removal, through DEAE-52 anion exchange column elution and collection 0.2 mol / L NaCl elution peak, and the uniform component obtained by Sephadex G-100 gel column chromatography purification.Its uronic acid content is 30%-40%, weight average relative molecular weight is 50-100kDa, mainly by glucose, galactose, galacturonic acid and arabinose, infrared spectrum has carboxyl characteristic absorption peak at 1736 cm-1 and 1615 cm-1.Experiments prove that the acanthopanax acid polysaccharide has antioxidant activity and hypoglycemic activity.The acid polysaccharide has protective effect on LPS-induced Caco-2 cell damage, can effectively reduce the secretion of pro-inflammatory factors TNF-α, IL-1β, IL-6, and improve the level of anti-inflammatory factor IL-10.The acanthopanax acid polysaccharide can be prepared into oral liquid, tablet candy, solid beverage and other functional food forms.

Description

Technical Field

[0001] This invention belongs to the field of functional food and biotechnology, specifically relating to an acidic polysaccharide extracted, isolated and purified from the rhizome of Acanthopanax senticosus, its preparation method and its application in the preparation of functional foods with intestinal protection, antioxidant and blood sugar lowering functions. Background Technology

[0002] Eleutherococcus senticosus is a plant belonging to the genus Eleutherococcus in the family Araliaceae. Its rhizome is the traditional medicinal part of the plant, possessing effects such as invigorating qi and strengthening the spleen, tonifying the kidneys and calming the mind. Polysaccharides are one of the main active ingredients of Eleutherococcus senticosus, and can be divided into neutral polysaccharides and acidic polysaccharides based on their structural characteristics. Among them, acidic polysaccharides, due to their rich uronic acid groups, possess unique physicochemical properties and stronger biological activities, such as anti-inflammatory and immunomodulatory effects, and have become a hot topic in functional food raw material research.

[0003] Caco-2 cells are a human colon adenocarcinoma cell line widely used as an in vitro model for studying intestinal barrier function. In vitro evaluation methods, such as cell experiments and biochemical experiments, have been widely applied in the screening of raw materials and functional evaluation of functional foods. Currently, there are no systematic reports on the use of in vitro experiments to evaluate the protective effect of Acanthopanax senticosus acidic polysaccharides on intestinal barrier function and to develop them into functional foods. Summary of the Invention

[0004] Purpose of the invention The purpose of this invention is to provide an acidic polysaccharide of Acanthopanax senticosus with well-defined physicochemical properties and biological activity. Another object of the present invention is to provide a method for preparing Acanthopanax senticosus acidic polysaccharide. Another object of the present invention is to provide the application of the aforementioned Acanthopanax senticosus acidic polysaccharide in the preparation of functional foods with intestinal protection, antioxidant and blood sugar lowering functions.

[0005] Technical solution To achieve the above objectives, the present invention adopts the following technical solution:

[0006] An acidic polysaccharide of Acanthopanax senticosus with intestinal protection function is characterized in that the acidic polysaccharide is a homogeneous component extracted and isolated from the rhizome of Acanthopanax senticosus, which is rich in uronic acid and has characteristic absorption peaks of carboxyl groups at 1736 cm⁻¹ and 1615 cm⁻¹ in infrared spectrum; the acidic polysaccharide is prepared by steps including anion exchange column chromatography and gel column chromatography.

[0007] Furthermore, the uronic acid content of the Acanthopanax senticosus acidic polysaccharide is 30% to 40%.

[0008] Furthermore, the weight-average relative molecular weight of the Acanthopanax senticosus acidic polysaccharide is 50 kDa to 100 kDa.

[0009] Furthermore, the weight-average relative molecular weight of the Acanthopanax senticosus acidic polysaccharide is 79 kDa.

[0010] Furthermore, the acidic polysaccharide of Acanthopanax senticosus is mainly composed of glucose, galactose, galacturonic acid and arabinose.

[0011] Furthermore, the anion exchange column chromatography is a DEAE-Cellulose-52 column chromatography, and the elution curve shows that the 0.2 mol / L NaCl elution fraction is the enrichment peak of the acidic polysaccharide.

[0012] Furthermore, the gel column chromatography is Sephadex G-100 gel column chromatography, and its elution curve shows a single symmetrical peak.

[0013] This invention also provides a method for preparing the acidic polysaccharide of Acanthopanax senticosus, characterized by comprising the following steps: S1: Take the rhizome of Acanthopanax senticosus, crush it, extract it with hot water, concentrate it, and precipitate it with ethanol to obtain crude polysaccharide of Acanthopanax senticosus; S2: The crude polysaccharide obtained in step S1 is deproteinized by the Sevag method and decolorized by AB-8 macroporous adsorption resin to obtain deproteinized and decolorized polysaccharide. S3: The polysaccharide obtained in step S2 was loaded onto a DEAE-Cellulose-52 anion exchange column and eluted sequentially with distilled water, 0.1 mol / L NaCl solution and 0.2 mol / L NaCl solution. The 0.2 mol / L NaCl eluent was collected to obtain the acidic crude polysaccharide of Acanthopanax senticosus. S4: The acidic crude polysaccharide obtained in step S3 is loaded onto a Sephadex G-100 gel column and eluted with ultrapure water or 0.1 mol / L NaCl solution. The main peak component is collected according to the elution curve, dialyzed, and freeze-dried to obtain homogeneous Acanthopanax senticosus acidic polysaccharide.

[0014] The present invention further provides the application of the Acanthopanax senticosus acidic polysaccharide in the preparation of functional foods with antioxidant functions, wherein the antioxidant functions include scavenging DPPH free radicals, ABTS free radicals, hydroxyl free radicals or chelating Fe²⁺.

[0015] The present invention also provides the application of the aforementioned Acanthopanax senticosus acidic polysaccharide in the preparation of functional foods with auxiliary hypoglycemic function.

[0016] The present invention also provides the application of the Acanthopanax senticosus acidic polysaccharide in the preparation of functional foods with intestinal protection function.

[0017] Furthermore, the intestinal protective function is to protect LPS-induced damaged Caco-2 cells by reducing the expression levels of TNF-α, IL-1β, and IL-6 in the cells and / or increasing the expression level of IL-10.

[0018] The present invention also provides a functional food composition, characterized in that it comprises an effective amount of the Acanthopanax senticosus acidic polysaccharide and a food-acceptable carrier or excipient.

[0019] Furthermore, the dosage form of the functional food composition is one of oral liquid, compressed candy, or solid beverage.

[0020] Beneficial effects Compared with the prior art, the present invention has the following beneficial effects: (1) The Acanthopanax senticosus polysaccharide obtained in this invention is an acidic polysaccharide with a higher uronic acid content compared to other polysaccharides. Moreover, the polysaccharide was isolated and purified from the rhizome of Acanthopanax senticosus by DEAE-Cellulose-52 anion exchange column chromatography and Sephadex G-100 gel column chromatography to obtain a homogeneous acidic polysaccharide, and the purification process was stable. (2) The acidic polysaccharide of Acanthopanax senticosus prepared by the present invention has antioxidant activity, blood glucose lowering activity and intestinal protection function. Experimental data show that it has the potential to be developed into a functional food. (3) The Acanthopanax senticosus acidic polysaccharide described in this invention can be prepared into various functional food forms such as oral liquid, compressed candy, and solid beverage. Attached Figure Description

[0021] Figure 1 This is the elution curve of the acidic polysaccharide of Acanthopanax senticosus of the present invention on a DEAE-Cellulose-52 anion exchange column.

[0022] Figure 2 This is a Sephadex G-100 gel column elution curve of the acidic polysaccharide of Acanthopanax senticosus in this invention.

[0023] Figure 3 This is a standard curve for the HPLC molecular weight determination of the acidic polysaccharide of Acanthopanax senticosus in this invention.

[0024] Figure 4 This is an HPLC chromatogram of the monosaccharide composition of the Acanthopanax senticosus acidic polysaccharide of the present invention.

[0025] Figure 5 This is a scanning electron microscope (SEM) image of the acidic polysaccharide of Acanthopanax senticosus in this invention.

[0026] Figure 6 This is the Fourier transform infrared (FT-IR) spectrum of the acidic polysaccharide of Acanthopanax senticosus in this invention.

[0027] Figure 7 This is a UV-Vis spectrum scan of the acidic polysaccharide of Acanthopanax senticosus in this invention.

[0028] Figure 8 This is a graph showing the DPPH free radical scavenging ability of the Acanthopanax senticosus acidic polysaccharide of the present invention.

[0029] Figure 9 This is a graph showing the ABTS free radical scavenging ability of the Acanthopanax senticosus acidic polysaccharide of the present invention.

[0030] Figure 10 This is a graph showing the hydroxyl radical scavenging ability of the Acanthopanax senticosus acidic polysaccharide of the present invention.

[0031] Figure 11 This is a graph showing the Fe²⁺ chelating capacity of the Acanthopanax senticosus acidic polysaccharide of this invention.

[0032] Figure 12 This is a diagram showing the inhibitory activity of Acanthopanax senticosus acidic polysaccharide against α-glucosidase.

[0033] Figure 13 This is a diagram showing the inhibitory activity of Acanthopanax senticosus acidic polysaccharide against α-amylase.

[0034] Figure 14 This is a graph showing the effect of Acanthopanax senticosus acidic polysaccharide of the present invention on the viability of Caco-2 cells (CCK-8 assay).

[0035] Figure 15 The figure shows the effect of different concentrations of LPS on the survival rate of Caco-2 cells.

[0036] Figure 16 This figure shows the effect of Acanthopanax senticosus acidic polysaccharide of the present invention on the expression of inflammatory factors in LPS-induced Caco-2 cells. Detailed Implementation

[0037] The present invention will be further described below with reference to specific embodiments.

[0038] Example 1: Preparation of acidic polysaccharides from Acanthopanax senticosus (1) Raw material pretreatment: Take dried Acanthopanax senticosus rhizomes, remove impurities, crush and pass through a 40-mesh sieve, and extract with 95% ethanol at 80℃ for 3 hours to remove small molecule impurities. The filter residue is used for later use. (2) Hot water extraction: Add the pretreated filter residue to deionized water at a material-to-liquid ratio of 1:15, extract at 90℃ for 3 hours, filter, collect the filtrate, repeat the extraction of the filter residue once, and combine the two filtrates. (3) Concentration and purification: The filtrate was concentrated to 1 / 4 of its original volume under reduced pressure at 60℃, and 4 times the volume of 95% ethanol was added to precipitate the precipitate. The precipitate was then allowed to stand overnight at 4℃. After centrifugation at 5000 rpm for 15 min, the precipitate was collected, washed successively with anhydrous ethanol and acetone, and dried to obtain the crude polysaccharide of Acanthopanax senticosus. (4) Deproteinization and decolorization: Prepare a 10 mg / mL aqueous solution of crude polysaccharide. Add 1 / 4 volume of Sevag reagent (chloroform: n-butanol = 4:1), shake vigorously for 30 min, centrifuge at 8000 rpm for 15 min, collect the upper aqueous phase, and repeat the operation 5-6 times. Collect the aqueous phase, concentrate it, and load it onto an AB-8 macroporous adsorption resin column. Elute with distilled water at a flow rate of 5 mL / min, collect the eluent, concentrate it, and freeze-dry it to obtain deproteinized and decolorized polysaccharide. (5) DEAE-Cellulose-52 column chromatography: Dissolve 2 g of the deproteinized and decolorized polysaccharide in an appropriate amount of distilled water and load the sample onto a DEAE-Cellulose-52 anion exchange column. Elute sequentially with distilled water, 0.1 mol / L NaCl, and 0.2 mol / L NaCl solution at a flow rate of 10 mL / min. Detect using the phenol-sulfuric acid method and plot the elution curve (e.g., ...). Figure 1 (As shown). The fraction corresponding to the elution peak of 0.2 mol / L NaCl was collected, dialyzed for 48 h, and freeze-dried to obtain the crude acidic polysaccharide of Acanthopanax senticosus. (6) Sephadex G-100 gel column chromatography: 20 mg of the above-mentioned acidic crude polysaccharide was dissolved in 2 mL of ultrapure water and loaded onto a Sephadex G-100 gel column. Elution was performed with 0.1 mol / L NaCl solution at a flow rate of 1 mL / min. The phenol-sulfuric acid method was used for detection, and an elution curve was plotted (e.g., phenol-sulfuric acid method). Figure 2 (As shown). Collect the single symmetrical main peak component, dialyze, and freeze-dry to obtain purified Acanthopanax senticosus acidic polysaccharide.

[0039] Example 2: Structural characterization of Acanthopanax senticosus acidic polysaccharides (1) Determination of uronic acid content: The uronic acid content was determined by the sulfuric acid-carbazole method and was 34.98%±2.97%. (2) Molecular weight determination: The weight-average relative molecular weight was determined by the HPGPC method and was 79 kDa. (3) Monosaccharide composition analysis: The PMP pre-column derivatization-HPLC method was used for analysis, and the results showed that it was mainly composed of glucose, galactose, galacturonic acid and arabinose. (4) Scanning electron microscopy analysis: such as Figure 5 As shown, the sample has an irregular sheet-like structure with a rough surface, wrinkles, and pores. (5) Infrared spectroscopy analysis: such as Figure 6As shown, carboxyl absorption peaks were observed at 1730 cm⁻¹ and 1620 cm⁻¹, confirming that it is an acidic polysaccharide. (6) Ultraviolet spectroscopy analysis: such as Figure 7 As shown, the sample has no obvious absorption peaks at 260 nm and 280 nm, indicating that there are no protein and nucleic acid impurities.

[0040] Example 3: Determination of the antioxidant activity of Acanthopanax senticosus acidic polysaccharides Different concentrations of Acanthopanax senticosus acidic polysaccharide solutions (0.2, 0.5, 1.0, 1.5, 2.0 mg / mL) were prepared, and their antioxidant activity was determined. The results are as follows: Figure 8-11 As shown, the scavenging ability of the sample against DPPH radicals, ABTS radicals, and hydroxyl radicals, as well as its Fe²⁺ chelating ability, were all concentration-dependent. At 2.0 mg / mL, the scavenging rates were 81.64±0.31%, 81.06±0.56%, and 85.40±0.92%, respectively, and the Fe²⁺ chelating rate was 52.18±0.48%.

[0041] Example 4: Determination of the auxiliary hypoglycemic activity of Acanthopanax senticosus acidic polysaccharides Different concentrations of Acanthopanax senticosus acidic polysaccharide solutions (0.5, 2.5, 5.0, 7.5, 10.0 mg / mL) were prepared, and their inhibitory activities against α-glucosidase and α-amylase were determined. The results are as follows: Figure 12-13 As shown, the inhibition rates of the samples against both enzymes were concentration-dependent, with IC50 values ​​of 2.3 ± 0.2 mg / mL and 3.8 ± 0.3 mg / mL, respectively.

[0042] Example 5: Protective effect of Acanthopanax senticosus acidic polysaccharide against LPS-induced Caco-2 cell damage (1) Cell viability assay: Caco-2 cells were seeded in 96-well plates and cultured for 24 h with different concentrations (6.25-200 μg / mL) of Acanthopanax senticosus acidic polysaccharide solution. Cell viability was then detected using the CCK-8 assay. The results are as follows: Figure 14 As shown, the samples were not toxic to Caco-2 cells in the concentration range of 6.25-200 μg / mL, and promoted cell proliferation at concentrations of 50 and 100 μg / mL. (2) LPS damage model establishment: Caco-2 cells were treated with different concentrations of LPS for 24 h, and 5 mg / L LPS treatment for 24 h was determined as the modeling condition (cell viability 73.89±2.70%). (3) Measurement of inflammatory factor levels: The experiment was divided into a blank group, a model group (5 mg / L LPS), and a polysaccharide protection group (50 μg / mL polysaccharide pretreatment + LPS). ELISA results showed that compared with the model group, the polysaccharide protection group had significantly lower levels of TNF-α, IL-1β, and IL-6, and significantly higher levels of IL-10 (P<0.05), indicating that the polysaccharide has a protective effect against LPS-induced Caco-2 cell damage.

[0043] Example 6: Preparation of Functional Foods (1) Preparation of health care oral liquid Take 50 g of the Acanthopanax senticosus acidic polysaccharide prepared in Example 1, add 600 mL of purified water and stir to dissolve. Separately, take 50 g of xylitol, 2 g of citric acid, and 2 g of potassium sorbate, dissolve them in an appropriate amount of purified water, and mix them. Add water to make up to 1000 mL, stir evenly, filter through a 0.45 μm filter membrane, fill into 10 mL glass bottles, and sterilize at 105℃ for 30 minutes to obtain the health care oral liquid. Each 10 mL bottle contains 500 mg of Acanthopanax senticosus acidic polysaccharide. (2) Preparation of compressed candy Take 100 g of the Acanthopanax senticosus acidic polysaccharide prepared in Example 1 and pass it through a 100-mesh sieve; add 200 g of sorbitol, 50 g of microcrystalline cellulose, and 5 g of magnesium stearate, and mix well. Directly compress the mixture into tablets using a rotary tablet press, adjusting the tablet weight to 0.8 g to obtain compressed candies. Each tablet contains approximately 230 mg of Acanthopanax senticosus acidic polysaccharide. (3) Preparation of solid beverages Take 100 g of the Acanthopanax senticosus acidic polysaccharide prepared in Example 1, add 300 g of maltodextrin, 150 g of erythritol, and 10 g of citric acid, and mix well. Add an appropriate amount of 90% ethanol to make a soft mass, granulate through a 14-mesh sieve, dry at 50°C for 3 hours, granulate through a 12-mesh sieve, and package into aluminum foil bags, 5 g per bag. Dissolve in warm water before drinking.

[0044] It should be noted that the preparation methods for the oral liquid, compressed candy, and solid beverage dosage forms described in Example 6 all employ conventional food processing techniques in the field, and the types and amounts of excipients involved are conventional choices in the field of functional foods. Those skilled in the art can make appropriate adjustments to the excipients according to actual production needs to achieve the preparation of the functional foods described in this invention.

Claims

1. An acidic polysaccharide of Acanthopanax senticosus with intestinal protective function, characterized in that, The Acanthopanax senticosus acidic polysaccharide is a homogeneous component extracted and isolated from the rhizome of Acanthopanax senticosus. It is rich in uronic acid and has characteristic absorption peaks of carboxyl groups at 1736 cm⁻¹ and 1615 cm⁻¹ in infrared spectrum. The Acanthopanax senticosus acidic polysaccharide is prepared by steps including anion exchange column chromatography and gel column chromatography.

2. The Acanthopanax senticosus acidic polysaccharide according to claim 1, characterized in that, The uronic acid content of the Acanthopanax senticosus acidic polysaccharide is 30% to 40%, and the weight-average relative molecular weight is 50 kDa to 100 kDa; preferably, the weight-average relative molecular weight is 79 kDa, and the Acanthopanax senticosus acidic polysaccharide is mainly composed of glucose, galactose, galacturonic acid and arabinose.

3. The Acanthopanax senticosus acidic polysaccharide according to claim 1, characterized in that, The anion exchange column chromatography was a DEAE-Cellulose-52 column chromatography, and the elution curve showed that the 0.2 mol / L NaCl elution fraction was the enrichment peak of the acidic polysaccharide; the gel column chromatography was a Sephadex G-100 gel column chromatography, and its elution curve showed a single symmetrical peak.

4. The method for preparing the acidic polysaccharide of Acanthopanax senticosus according to any one of claims 1-3, characterized in that, Includes the following steps: (a) After crushing the rhizome of Acanthopanax senticosus, extract it with ethanol by reflux to remove small molecule impurities, and keep the filter residue for later use; (b) Add the pretreated filter residue to deionized water at a material-to-liquid ratio of 1:10-1:20, extract at 80-100℃ for 2-4 hours, filter, and collect the filtrate. (c) After concentrating the filtrate under reduced pressure, add ethanol to precipitate the precipitate, collect the precipitate, and dry it to obtain crude polysaccharide; (d) After dissolving the crude polysaccharide, the sample was loaded onto a DEAE-Cellulose-52 anion exchange column and eluted with a gradient of 0.1-0.5 mol / L NaCl solution. The eluted fraction with 0.2 mol / L NaCl was collected. (e) The fraction obtained in step (d) was loaded onto a Sephadex G-100 gel column, eluted with 0.1 mol / L NaCl solution, and the homogenized fraction was collected and freeze-dried to obtain purified Acanthopanax senticosus acidic polysaccharide.

5. The application of the Acanthopanax senticosus acidic polysaccharide according to any one of claims 1-3 in the preparation of functional foods with antioxidant functions, characterized in that, The antioxidant function includes scavenging DPPH free radicals, ABTS free radicals, hydroxyl free radicals, or chelating Fe²⁺.

6. The use of Acanthopanax senticosus acidic polysaccharide according to any one of claims 1-3 in the preparation of functional foods with auxiliary hypoglycemic function.

7. The application of the Acanthopanax senticosus acidic polysaccharide according to any one of claims 1-3 in the preparation of functional foods with intestinal protection function, characterized in that, The intestinal protective function is to protect LPS-induced damaged Caco-2 cells by reducing the expression levels of TNF-α, IL-1β, and IL-6 in the cells and increasing the expression level of IL-10.

8. A functional food composition, characterized in that, It includes an effective amount of the Acanthopanax senticosus acidic polysaccharide as described in any one of claims 1-3, as well as a food-acceptable carrier or excipient.

9. The functional food composition according to claim 8, characterized in that, The dosage form of the functional food composition is an oral liquid, compressed candy, or solid beverage.

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